Cystic fibrosis (CF) is the most common genetic disease in Caucasian populations, with an incidence of 1 in 2,000 live births and a carrier frequency of approximately 1 in 20. It is inherited as an autosomal recessive disease. The cystic fibrosis gene has been mapped, cloned, and sequenced (Ronimens et al., Science 245:1059-1065 (1989); Kerem et al., Science 245:1073-1080 (1989)). The gene product is the cystic fibrosis transmembrane regulator (CFTR) which functions as a chloride ion channel in the apical membranes of secretory epithelial cells (Anderson et al., Science 251:679-682 (1991)). The expression of the CFTR is most prominent in sweat glands and the respiratory and gastrointestinal tracts (Collins, F. S., Science 256:774-779 (1992)). CF is a disease of the epithelial cells, and the distribution of CFTR is essentially consistent with the clinical pathology.
Cystic fibrosis (CF) is caused by any one of a thousand mutations that affect the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR produces chloride (Cl−) channels that are regulated by cAMP. These channels are expressed in many cellular epithelia including the lung, pancreas, intestine, hepatobiliary tract, sweat gland, and vas deferens (Sheppard, D N and Welsh M J. Physiol Rev 79: S23-S45, 1999). The CFTR gene was cloned in 1989 (Riordan J R, et al. Science 245: 1066-1073, 1989).
In CF, the functionally defective apical membrane chloride channel secondarily leads to a loss of luminal sodium and water. In airways, the increase in sodium absorption and reduction in chloride secretion both lead to a loss of airway surface water. Thus, airway mucus is thickened because of insufficient endobronchial water. Bronchiolar plugging and decreased mucociliary clearance follow. These changes in the pulmonary environment result in an increase in bacterial colonization. The colonization of the lung leads to a cycle of inflammation, destruction, and further colonization. The microorganisms colonizing the lungs attract neutrophils which contain and resolve a pulmonary infection in the normal host. However, in the patient with CF, the release of proteolytic enzymes by neutrophils further damages lung parenchyma. Neutrophils release neutrophil elastase which causes many typical pathologic features of CF, including epithelial damage, bronchial gland hyperplasia (leading to increased mucus production), and connective tissue damage resulting in bronchial distortion (Stockley et al., Clin. Sci. 74:645-650 (1988)). Neutrophils also release serine protease, a factor that damages bronchial cilia (Cole, P. J., Eur. J. Respir. Dis. 69(suppl 147):6-15 (1986)). The damaged bronchial cilia have decreased ciliary beat frequency and a reduction in mucociliary clearance. In the normal host, neutrophil proteases are controlled by the naturally occurring protease inhibitors found in the pulmonary tree. The most potent of these, alpha-1-antitrypsin, irreversibly binds with high affinity to serine protease. However, in the lung of the patient with CF, proteases are produced by a variety of cells besides neutrophils, including pulmonary macrophages and the microorganism, Pseudoinonas aeruginosa (Fick et al., Chest 95:215S-216S (1989)). This excessive protease production overwhelms the naturally occurring endogenous protease inhibitors.
As a result of these physiological changes, respiratory tract diseases are responsible for more than 90% of the morbidity and mortality in CF (Hata et al., Clin. Chest Med. 9:679-689 (1988)).
In the airway, defective CFTR impairs Cl− secretion from epithelial cells and increases their sodium (Na+) absorption. This changes the normal ion composition and dehydrates the airway surface liquid (ASL), thus decreasing its volume. Airway mucus becomes thick and is poorly cleared from the lungs, which enables bacteria to colonize the area. Eventually respiration fails (Verkman A S, Song Y and Thiagarajah J R. Am J Physiol Cell Physiol 284: C2-15, 2003).
There has been considerable work to repair CFTR's Cl− ion transit deficiency, but rescue has not been practical so far. For example, the most common CFTR mutation ΔF508-CFTR is amenable to rescue at 27° C., but that temperature is too low for the human body. Some chemicals have shown that they can rescue CFTR, and the best are under Phase I or Phase II clinical trials.
Several non-CFTR chloride channels have been identified in airway cells including the family of voltage-dependent Cl− channels, volume-regulated anion channels (VRAC), and, calcium (Ca2+)-activated Cl− channels (CaCCs) (Nilius B and Droogmans G. Acta Physiol Scand 177: 119-147, 2003; Verkman A S, Song Y and Thiagarajah J R. Am J Physiol Cell Physiol 284: C2-15, 2003). Previous studies have shown that CaCCs are present in the apical membrane of both normal and CF airway epithelia (Anderson M P and Welsh M J. Proc Natl Acad Sci USA 88: 6003-6007, 1991). Even in CF, Ca2+-activated Cl− secretion is intact and provides a therapeutic target to circumvent the Cl− secretion defect in CF (Nilius B and Droogmans G. Acta Physiol Scand 177: 119-147, 2003). Denufosol and Moli1901, which stimulate CaCC and target this therapeutic pathway, are currently being evaluated in clinical trials. Denufosol, a chemically stable P2Y2 receptor agonist, was shown to increase chloride and fluid secretion in preclinical studies (Kellerman D, et al. Pulm Pharmacol Ther 21: 600-607, 2008). It improved the lung function by 2.5% in a 24 week treatment in a phase III trial (Storey S and Wald G. Nat Rev Drug Discov 7: 555-556, 2008). Moli1901 (formerly known as Duramycin) is a stable polypeptide. In a phase I trial, Moli901 stimulated Cl− transport in normal and CF epithelia (Zeitlin P L et al. Chest 125: 143-149, 2004). 4 weeks of treatment with aerosolized Moli1901 caused a 2% increase in lung function (Storey S and Wald G. Nat Rev Drug Discov 7: 555-556, 2008). Although these two agents are being studied intensively, the discovery of more compounds to activate this pathway without any adverse effects would still be beneficial for the potential treatment of CF.
High-throughput screening allows assessment of a large number of compounds quickly, and has been used widely in drug discovery in recent years (Monteith G R and Bird G S. Trends Pharmacol Sci 26: 218-223, 2005). A goal was to search the 2000 compounds of MicroSource Discovery's MSSP library for compounds that would enhance cytoplasmic Ca2+, which in turn would activate the CaCCs and alleviate the defective Cl− secretion in CF airways without adverse side effects.
It has been previously disclosed that zinc with ATP or zinc alone reliably causes a sustained increase in cytoplasmic Ca2+ in cells bathed in a Ringer's solution modified to enhance Ca2+ transfer (ECaT Ringer's see Table 1). Both zinc and ATP activate P2X purinergic receptors that are non-selective cation channels expressed in human airway surface epithelia (Liang L, et al. Am J Physiol Cell Physiol 289: C388-C396, 2005; Zsembery A, et al. J Biol Chem 278: 13398-13408, 2003; Zsembery A, et al. J Biol Chem 279: 10720-10729, 2004). The resulting increase in cytoplasmic Ca2+ then translates into sustained Cl− secretion in both CF and non-CF airway epithelia in vivo and in vitro. Zinc, however, is a biometal, so the amount of it taken into the body would be a concern in CF therapy.